Cell Culture - Science topic

Dear Malcolm.

Thank you so much for your response and so well-detailed advice and suggestions.

I really appreciate.

Hello Akshaya Nagarajan

It is not contamination.

From the pic it seems clear that the cells are seeded at a high density. U-87MG cells are known for their tendency to aggregate and form clusters, especially when cultured at high densities. The presence of these structures can affect the overall morphology and uniformity of the culture, potentially impacting experimental results.

To minimize the formation of these clusters, you may reduce the seeding density. You may seed U-87MG cells at a lower initial density, at 1 x 10^4 cells/cm2. During passaging, you may remove the clusters by waiting for them to settle to the bottom of the flask, and then carefully aspirating the supernatant containing single cells.

Best,

Thank you, Malcolm Nobre and Souradip Sinha for your insights. As suggested, I had split the cells and seeded them in a lower density, which seems to have reduced the clusters by a considerable amount. However, compared to other cancer cell line at my disposal, only these are growing over each other.

Poulami Ghosh

Hi Poulami,

By background, do you mean contamination - either bacterial or other cell types (fibroblasts etc.)?

Suggestions:

Hi, Erica Weber.

It is not recommended to perform ELISA testing through specific targets because the immunogen is a "wild-type cancer cell that naturally expresses multiple genes".

You can try to purify the supernatant with Protein A/G Magnetic Beads, and then perform SDS-PAGE purity identification + A280 detection concentration.

MCE provides reliable Protein A/G Magnetic Beads (HY-K0202), which has the following advantages:

1. Only a small amount of magnetic beads is required for immunoprecipitation.

2. Convenient and time-saving.

3. Low non-specific binding rate.

4. Less sample loss.

5. Protein binding capacity is as high as 0.7 mg/mL.

6. Stable.

The specific operation process can be found on the MCE official website (https://www.medchemexpress.com/inhibitor-kit/protein-a/g-magnetic-beads.html).

The process directory is as follows:

1. Antigen sample preparation.

2. Magnetic bead pretreatment.

3. Antibody binding to magnetic beads.

4. Antigen binding to antibody-magnetic bead complex.

5. Antigen elution.

Thank you so much Lina, i will give it a try Lina M. Yanygina

Hi, Did you try something from Sensair as a replacement for the fyrite system. How is it working out? We are also currently looking for alternatives for fyrite to check our incubator 5% CO2 levels. Thank you. Rebecca

Thank you so much Malcolm Nobre

Hello Hayato Saiki, unfortunately I couldn't figure out what could have happened. The problem continues if I thaw some of the cells from the same era. I imagine that it may be some kind of contamination, by viruses, or something like that, and not associated with some reagent such as culture medium, fetal bovine serum, for example. Recently I found a very old vial of C2C12 that I was able to obtain the same speed of growth and differentiation stimulus as those I obtained in the past. My advice is to try to thaw as many vials as possible, and keep trying.

Malcolm Nobre Thank you for your attention and for your thoughtful response to my observations. The size and appearance of the structures suggest that it might indeed not be a contamination. However, their movement (at least to me) does not seem to be Brownian, but rather directional. I have previously observed small, dark, motile structures — which I interpreted as contamination — using this same observation method with Trypan Blue. Some of my colleagues also interpreted these structures as indicative of contamination. In any case, I will keep these cultures and perform experiments to determine whether they are contaminated or not, and I will surely keep your advice in mind.

Thank you for the reply Alwyn Dart. We did a troubleshooting experiment with just the media for a couple of days. But we didn’t find anything.

Hello! I am wondering if you were able to pinpoint the cause of your culturing issue. I am currently having the same troubles.

Good morning,

I am wondering if you figured out the source of your culturing issues (specifically after passaging), as I am having the same issue!

Try to stain with DAPI or PI to see if any of the dots stain as nucleated material. And check the contamination on a fungal growth medium ( like PDA, Corn meal Agar, Czapek Dox agar). LB is more suitable for bacterial contaminations, maybe you have some (yeast cells/spores)

Hi Asghar Mohammadzadeasl

Hope all is well. May I ask if you have had any luck on your end finding these? We have been looking for it.

Apart from the suggested supplements, EGF, cholera toxin, hydrocortisone, and even insulin are usually required for the MCF10 cells to grow and survive in EMEM media since optimized media for epithelial cells is DMEM. By closely monitoring the attachment, morphology and proliferation rate, you would be able to find at which stage the cells are dying and add the additives accordingly.

The protocol for measuring cell viability using the Viability/Cytotoxicity Assay Kit for Live & Dead Cells (Calcein/PI) (MCE, HY-K1094) is attached below for your reference.

Since staining conditions vary depending on cell type and cell concentration, it is recommended to perform pre-experiments to determine the optimal concentrations of Calcein-AM and PI staining solutions.

1. Dead Cells preparation: Incubate cells in 70% ethanol for 30 min or in 0.1% saponin for 10 min.

Note: The method for preparing dead cells is not fixed. Alternative methods, such as treatment with 0.1%-0.5% digitonin for 10 min, can also be used.

2. Reagent Preparation: Remove the PI and Calcein-AM staining solutions to equilibrate at room temperature for 30 min.

3. PI Staining: Prepare several concentrations of PI working solutions (0.1-10 μM) using the assay buffer and stain the dead cells. Identify the concentration of PI that stains the nuclei without staining the cytoplasm.

4. Calcein-AM Staining: Prepare several concentrations of Calcein-AM working solutions (0.1-10 μM) using the assay buffer and stain dead cells. Identify the optimal Calcein-AM concentration that does not stain the cytoplasm. Subsequently, use this concentration to stain live cells and verify that live cells can be successfully stained.

Note: It is generally recommended to use the lowest possible dye concentration that provides sufficient signal intensity for the experiment.

The recommended staining concentrations for PI and Calcein-AM are 0.1–10 μM. The optimal concentration can be determined based on pre-experimental results.

The following example uses 2 μM Calcein-AM and 8 μM PI:

1. Reagent Preparation: Remove the PI and Calcein-AM staining solutions and equilibrate at room temperature for 30 min.

2. Preparation of Staining Working Solution: Add 5 μL of PI staining solution and 5 μL of Calcein-AM staining solution into 10 mL of assay buffer. Mix thoroughly to obtain the staining working solution, which can be used directly for staining.

Adherent Cells

1. Seed adherent cells into cell culture plates, microplates, or prepare cell coverslips.

Note: Suspended cells can also be prepared as cell coverslips.

2. After treating the cells according to the experimental design, wash the cells 2–3 times with PBS to completely remove residual active esterases in the culture medium.

3. Add an adequate amount of staining working solution, ensuring that the monolayer cells are fully covered.

4. Incubate at 37°C for 15 - 30 min.

Suspended Cells

1. After treating the cells according to the experimental design, centrifuge at 1,000 rpm for 3 min, discard the supernatant, and collect the cells.

Note: Recommended cell quantity is 1 × 104 - 1 × 105.

2. Wash the cell pellet 2 - 3 times with PBS to completely remove residual active esterases in the culture medium.

3. Resuspend the cell pellet in 100 μL of staining working solution, ensuring a cell density to 1 × 105 - 1 × 106 cells/mL。

4. Incubate at 37°C for 15 - 30 min.

Fluorescence Microscopy Detection

1. Adherent Cells: For cells in culture plates, aspirate the staining working solution to stop the staining. Wash the cells 2-3 times with PBS, and add a sufficient amount of assay buffer to completely cover the monolayer cells for observation. For cell coverslips, add 10 μL of assay buffer to a clean microscope slide to fully cover the coverslip for observation.

Note: Cell coverslips can be sealed with nail polish to prevent evaporation.

2. Suspension Cells: Add 10 μL of the stained cell suspension to a clean microscope slide. Seal with nail polish to prevent evaporation.

3. Detection: Use a fluorescence microscope with an excitation wavelength of 490 ± 10 nm to simultaneously observe live cells (yellow-green fluorescence) and dead cells (red fluorescence). Additionally, use an excitation wavelength of 545 nm to observe dead cells alone.

Fluorescence Microplate Reader Detection

1. Control Setup: Prepare control and experimental groups following the staining working solution preparation and staining steps.

Experimental Groups: A, B

Control Groups: Dead Cell Controls (C, D), Live Cell Controls (E, F), and Cell-Free Controls (G, H).

Note: Dead cell control can refer to the pre-experimental dead cell preparation method.

2. Adherent Cells: Can be directly detected.

3. Suspension Cells: Seed 100 μL of the stained cell suspension per well in a microplate.

Note: The minimum detectable cell count per well is 200 - 500 cells, and the maximum is 1 × 106 cells.

4. Detection: Configure appropriate excitation and emission wavelengths to collect data.

For Calcein-AM: Excite at 490 ± 10 nm and collect emission signals at 530 ± 12.5 nm.

For PI: Excite at 530 ± 12.5 nm (typical Rhodamine optical filter) and collect emission signals at 645 ± 20 nm.

Note: It is recommended to use a microplate reader with optical filters to ensure signal interference is minimized.

5. Analysis and Calculation

Define the percentage of live and dead cells based on fluorescence readings.

Absolute live and dead cell counts: Generate standard curves of cell counts versus fluorescence readings (530 nm and 645 nm). Fluorescence intensity correlates linearly with cell count in the sample.

Note: Dead cells exhibit strong signals at 645 nm and weak signals at 530 nm.

Flow Cytometry Analysis

For both suspension cells and trypsin-dissociated adherent cells, follow the above staining working solution preparation and staining steps. The stained cell suspension can be directly analyzed using flow cytometry.

Thanks a lot

hai Malcolm Nobre thank you very much for your answer, this answer so helpful for me~

i hope you always stay health

Hi Geetha,

I know you chose the hTERT-RPE-1 cell line to visualize cilia for obvious reasons. I also think this problem is not that much during siRNA transfection. This problem of transfecting plasmids, regradless of its size, is always there with the cell line. I also faced similar issues even while transfecting empty-GFP vectors. The exact reasons I’m not aware of, but possibly due its endogenous modification to make this terminally differentiated cell a propagative one.

What you can do to troubleshoot the condition is to use a very good quality RPE-1 cells and check transfecting some empty vectors first. Even before that you can keep your cells with only lipofectamine-3000 (without any plasmid or siRNA) to check whether the reagent that you're using is free of contamination and not causing the stress. Try to maintain the cell quality, else even after successful transfection you'll not be able to observe cilia (too much stress hinders with cilia formation in the cells).

The *other things" observed could,

1. Protein precipitation which can be caused by the high temperature ~30°C. At this temperature, protein can undergo cellular precipitation.

2. Precipitation of supplement items due to improper mixing

3. It could be extra cellular vesicles or some apoptic cell , which can be caused by cellular stress due to the temperature

4. It night also be plastic fragments from plastic wares

Suggestion

Try microscopic examination to evaluate the presence of absence of mycoplasma and dormant fungi.

This question lies outside my profession.

Hello Mahe Jabeen ,

It looks like cell debris (chunks of dead cells) to me.

If you have fewer dead cells, you'll have less debris. Some cell death during differentiation is quite normal (the Stem cell population is never homogeneous), but if it is excessive, you might consider doing some quality checks for your stem cell line.

I have also got the same result but not by immunoprecipitation, in my western from the plant tissue where I did western from endogenous protein, I am getting ~47kDa band, but the actual size of my protein of interest is ~43kDa (~389aa). Is it possible that the protein of interest in my case is going any PTM?

I would like to add information about the technique of chromosome preparation in vivo, though this technique is not applicable to tissue culture or in vitro. However, researchers can follow this technique to standardise the chromosome preparation in vitro conditions. A good, condensed, deeply stained, and well-spread chromosome is essential to constructing a karyotype.

Chromosome preparation from bone marrow cells of mice or rats or even in tumour-bearing mice in vivo was performed by following the mitotic division inhibition technique (Chakrabarti et al., 1985; Mallick et al, 2018; Banerjee et al, 2019; Chowdhury and Banerjee, 2020;21). The technique is brief: i) Specimens received an injection of 0.04% colchicine solution intraperitoneally at a rate of 1 ml/100 g body weight for 1 hour 30 min. ii) Cells from bone marrow were collected in hypotonic solution (0.075MKCl) and aspirated. Then, the cell suspension was incubated for 30 min at 37 °C and centrifuged for 10 min at 1500 rpm to collect the cell sediment. The sediment was fixed in aceto-alcohol fixative (3:1, methanol: glacial acetic acid, v/v) and centrifuged for 8 min. The sediment was fixed again and kept for chromosomal slide preparation. The flame-dried chromosome-prepared slide was stained with 5% Giemsa diluted in phosphate (pH - 7) buffer for 50 min. Then slides were washed in running tap water. Well-spread metaphases were observed and analysed under the Binocular Research Microscope (10 100 magnifications) to construct karyotypes for studying chromosomal aberrations.

In general it can, but the quality will be slightly worse than on a standard biological inverted microscope.

why not deliver the recombinase as mRNA - it is done for fish transgenesis. The transposase can be transcribed from a plasmid template using an in vitro transcription kit. The mRNA is co-injected with the plasmid. Here you would have to lipofect the mRNA later to remove the transgene

Hola Diego! Actualmente tengo el mismo reto, quiero precipitar o concentrar las proteínas de mi sobrenadante celular para después hacer Western Blot, pudiste hacerlo con TCA?

Hi, Bushra!

The cells may be contaminated with mycoplasma, so it is recommended to perform mycoplasma contamination-related tests. Additionally, hypoxia could also be a possible cause. It is advisable to check the culture conditions in the incubator for further observation.

Hope this helpful. Feel free to reach out for more details.

From the image you presented, the primary neuron seems to have partial aggregation, indicating some issues happened during primary culture preparation. Like Elliot said, the swelling structures along the dendritic shaft of a neuron are typically referred to as dendritic varicosities or dendritic swellings. These structures can have different functional and pathological implications, depending on the context. Small, bulbous protrusions along the dendrite, involved in synaptic transmission and primary site of excitatory synapses. These enlargements along the dendritic shaft can be transient or stable, sometimes forming en passant synapses (synapses along the shaft rather than at spines) during synaptic remodeling, or in response to activity. Moreover, neurodegenerative conditions (e.g., Alzheimer’s disease, Parkinson’s disease) can lead to dystrophic dendrites, characterized by abnormal swellings. Hypoxic/Ischemic damage can cause dendritic beading, an indicator of neuronal stress. Excitotoxicity due to excessive glutamate can also lead to dendritic swelling. The feather-like structure is difficult to say anything under this staining. At least the MAP2 neural marker and DAPI counter stain should be performed for the further investigation.

These are mycoplasma. They show movement, there numbers are huge. If you trypsinize and centrifuge at low speed, you will pellet down cells. But these particles remain suspended. You can put these I a well and observe under microscope.

Hello

No . We see it with lot of different epethilial as well as cancer cell lines

This is a picture of just the contamination.

Thank youfor your reply :)

To prepare 50 mL of a 2.5 mM 2-DG solution, given that the molecular weight of 2-DG is 164.16, the amount of 2-DG required (in mg) can be calculated by multiplying the concentration (mM), volume (mL), and molecular weight, which equals 20.52 mg. It is highly recommended to use online tools, such as molar calculators, where you can directly input the desired concentration and volume of the solution, and the calculator will provide the required mass of 2-DG powder. For example, if 1 g of 2-DG is dissolved in 1 L of culture medium, the resulting concentration would be a 6.09 mM solution

Hi Omama,

The MTT or CCK8 assay can be directly performed in a 96-well plate. Alternatively, you could also culture cells in a 6-well plate, followed by trypsin digestion and transfer to a 96-well plate for the MTT assay. This is theoretically feasible. However, both trypsin digestion and MTT assay can affect cell viability. Therefore, after trypsinization, it is recommended to let the cells recover for 2-6 hours until they fully adhere, before adding the MTT reagent.

Here is a reference MTT assay protocol for your consideration:

1. Preparation of MTT working solution

Dissolve MTT powder in PBS to prepare a 5 mg/mL MTT solution.

2. Cell proliferation assay (96-well plate)

2.1 Cell seeding: Prepare a single-cell suspension in culture medium containing 10% FBS, and seed cells into a 96-well plate at a density of 1,000 to 10,000 cells per well, with a total volume of 100 μL per well.

2.2 Cell culture: Incubate at 37°C, 5% CO₂ for 24 to 72 hours.

2.3 Adding MTT: Add 10 μL of MTT solution to each well, incubate for 4 hours, then remove the supernatant. For suspension cells, centrifuge first before removing the supernatant.

2.4 Dissolving formazan crystals: Add 100 μL DMSO to each well, and shake for 10 minutes to fully dissolve the crystals.

2.5 Measuring absorbance: Measure the absorbance at 562 nm using a microplate reader to assess cell proliferation.

This protocol is provided by the MCE Technical Team Binbin Lee — hope you find it helpful!

Hello Kristen Navarro

The molecular weight of 2-Deoxy-D-glucose is 164.16g/mol. So, one gram molecular weight (= 1 mole) is 164.16g. If you dissolve 164.16g of 2-Deoxy-D-glucose in a final volume of 1litre, you will have a 1M 2-Deoxy-D-glucose solution.

To get a 1M 2-Deoxy-D-glucose solution in a final volume of 50ml, you will have to weigh 8.2g of 2-Deoxy-D-glucose.

So, to get 50mM 2-Deoxy-D-glucose in a final volume of 50ml of DMEM media, you will have to weigh 0.41g (410mg) of 2-Deoxy-D-glucose powder and dissolve in 50ml DMEM media.

Best,

Hi,

Most of your iPSC colonies do not have the correct morphology. In my opinion, they folded and formed spontaneously EBs. Because the plate was covered with ECM, the folded colonies had adhered to the plate. Probably the iPSC colonies are floating too long in the falcon, which gives them time to fold before plating.

To get the morphology of iPSC colonies correct:

1. Passage the iPSC colonies more often when they are not yet connected.

2. Use methods of passaging that do not require centrifugation.

3. Spread the iPSC colonies quickly onto new plates.

4. Immediately spread the iPSC colonies onto new plates during passage in a ratio of 1:6 or 1:12 with droplets.

Good luck!

I have refilled numerous Fyrites over the years and always used 0-20% solution. However, I believe the solution would work in a Fyrite that is graded from 0-7%. The only issue i can see is that if your incubator has for example 8% CO2, which is required for suspension HEK cells, then your Fyrite could not measure this level accurately. Concerning an earlier comment, I would never rely on the CO2 level which is indicated by the incubator without first calibrating the reading first using a Fyrite. I have seen many times incubators indicating 5% CO2 which after testing with a Fyrite turned out to be way less or way more. When cells start growing strangely in a CO2 incubator, the first thing I do when troubleshooting is to measure the CO2 level with a fyrite.

Hello Elif Öztemiz Topcu

The use of cell scrapers is actually a harsh method to detach cells from the substratum. It does cause plasma membrane breakage and cell death. Due to cell death, free DNA from dying cells is released in culture media. The sticky nature of DNA causes cells and other debris to aggregate into large clumps.

You will have to remove the DNA released by dead cells by including 25-50 µg/ml DNase I in the presence of at least 1mM Mgcl2 (DNase I requires a concentration of at least 1mM Magnesium to work effectively) in your cell suspension as well as prevent cation dependent cell-cell adhesion by adding EDTA at a concentration of up to 5mM.

Best,

I believe that complete RPMI-1640 (10% FBS, Pen-Strep-Gen, enriched with mercaptoethanol should do the trick. DMEM would be an option as well.

very difficult to see from a picture; with yeast sometimes you see bourgeonning figures ...

Hello Bruno Costa,

You may refer to my answer in the post below.

Are you following these steps in your freezing and thawing protocol?

Best,

Dear Angel Cabrera Pereira ,

To my knowledge Glutamine is the only substance that might age (decay). If you would add additional Glutamine it should be fine (at least for basic cell culture).

Best wishes

Soenke

Hello Sayani Das,

I have experience in culturing the primary shrimp cells collected from some organs, such as the heart, muscle, and androgenic glands. I had the same issues with debris. In my opinion, there are some potential reasons.

1- The contamination of mycoplasma. If cells were isolated from fish gonads (primary cells), it was very easy to get mycoplasma infection. PBS or HBSS cannot help you solve this problem.

=> You can use the mycoplasma test kit to check. If there is a positive result with mycoplasma infection, you can use a Mycoplasma Removal Agent to add to the cell culture medium. The drawback is the cell growth rate might be reduced.

BTW, to reduce the contamination, you should sterilize your tools carefully before experimenting, wash the organs many times in the PBS + antibiotics, and culture the isolated cells in the cell culture medium + antibiotics.

2- I'm unclear whether your cells are primary or cell lines. If they are primary cells, they will easily die after several days of culture because they are not stem cells. The debris might be from this reason.

3- It might be that the cell culture medium is unsuitable for this cell type, so the cells might have died. You should be careful when checking the cell culture medium's salinity, pH, and osmotic condition. Your cells are fish cells, these conditions will affect a lot on the cell health.

Hope that my experience is helpful to you.

Check the media, pipettes, PBS, Trypsin and any other tools can be used ..use new

Hello Naile Merve Guven,

In order to estimate the protein concentration from HeLa lysates, you would need to perform a colorimetric biochemical assay such as bicinchoninic ad assay (BCA) or Bradford. Compare your sample OD values with that of the standard BSA samples in order to determine the test sample concentration. To further confirm if your protein concentration is good enough, you can refer to the product manuals or literature. Although this will only help to have an idea about a standard specific range, can vary as per alterations of your experiment conditions.

Hi Kyutae,

I cannot comment on the expression of your protein, but can say that proteins are indeed stable at -70C. Problems can arise during the freezing and thawing processes when proteins can concentrate and aggregate. Snap freezing can help minimize issues, but may not be enough for your protein. You can try formulating the solution with something like trehalose. I've attached a review article on formulation that can give a more complete picture, I'm definitely not a formulation expert. Good luck!

These look like syncytium (multinucleate cells). Could be at P7 that your cell population is starting to become senescent And not dividing properly.

Yes, the first 2 images look like the plasti. Maybe the outside of the culture flask. The 3rd image has some out of focus material that could be groups of cells.

Hello Juha Park

You may consider the following.

1. RAW 264.7 cells will self-activate when grown to high density before splitting. Keeping them at lower density should stop self-activation. Cells should be split when the confluency is between 70 and 80%.

2. During subculture, do not use trypsin for cell detachment. RAW 264.7 cells are sensitive to external stimuli. Trypsin can activate these cells. Trypsin treatment may greatly influence the cell membrane protein composition and reactivity of the cell to media components. Instead, you may use EDTA, manual scraping, or a mixture of the two to harvest RAW 264.7 cells.

3. Test your cells for mycoplasma contamination.

4. Have you changed the lot/brand of FBS used in the media? The serum can have lot-to-lot effects.

FYI: The standard media used for the growth and maintenance of RAW 264.7 cells is DMEM. Though RPMI-1640 has been used to maintain RAW 264.7 cells, there are a few differences in the composition that you should make a note of.

For instance, RPMI-1640 lacks the high level of glucose present in DMEM (4.5g/L) which in turn could affect the response of RAW 264.7 to external stimuli.

You may want to refer to the paper attached below for more information.

Best.

Hello Louise Piilgaard Petersen ,

Based on your description and the figures, the amphotericin B did not cause either growth inhibition of apoptosis of the component in your culture medium, and the component seems in the different layer other then cells, which I observed that was out of focus in the microscopic image, I suggest this contamination suspects is an artificial object such as polypropylene fiber from the tips of pipette. Therefore, there should be no contamination in your cell sample.

Best

Hi David Schad

I'm lysing my transwell cultures for proteomics, but my method may still give you some ideas.

I'm using the 12mm transwells, after washing in PBS I peel the membrane from the support (could also be cut out). I then put half a membrane (or a whole 6.5mm membrane) in the bottom of a 1.5mL Eppendorf tube, and store them dry at -80.

When it comes time to lyse my cells (using 8M urea in my case), I just add the volume of lysis buffer to the Eppendorf and you can lyse on a rocker overnight, sonicate, vortex, or what ever works best for your type of cells. No problems with lysis buffer seeping through the membranes, because it's all contained in the Eppendorf.

Best of luck!

Sam

Hi Elisabetta,

Please check our newest work. I hope it helps you and solves your questions.

This is not quite what I need. I want to make a media from the pancreas by grinding the pancreas up and removing enzymes and cell debris. I want to culture my cells in a media that is made from pancreas.

Hi Bjarte Skoe Erikstein , thank you for answering my question!

If i understand it right, then high pH Value could increase production of molecules which then interact with the resazurin. the high viability values are more likely "false positive" as they show cell stress, right?

In which way does Resazurin interact with ROS? More ROS = high Resazurin reaction? Or is it the other way around? Normally my cells are not showing a toxic effect from the resazurin in my controls, but in the higher pH value samples the cells looked disturbed under the microscope. so values above 140% "viability" are more likely a sign of mitochondrial stress, right?

Hello Echo Zhu,

The use of high trypsin concentration may have significantly reduced the cell's ability to form adhesive bonds with adsorbed cell adhesion proteins by decreasing the number of functional integrins available on the cell membrane.

You may prevent integrin damage by using either low trypsin concentration or reducing digestion time from 30 minutes to 10-15 minutes with periodic shaking, which may result in substantially improved cell adhesion.

Best.

Looking at the third picture, I would say yes they are contaminated.

Hello Faezeh Molaei

Poor aqueous solubility causes precipitation from aqueous media after dilution of DMSO stock solution. Hesperetin is soluble in DMSO but may not be soluble in DMSO-aqueous mixture. The polarity of DMSO is not the same as that of the mixture (DMSO-aqueous media). You may solve this problem by adding a carrier like cyclodextrin.

Enhanced water solubility of hesperetin can be achieved by complexing with either D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) or phosphatidylcholine (PC). So, you may prepare Hesperetin-TPGS micelles or hesperetin-PC complexes, instead of using free Hesperetin. Hesperetin-TPGS micelles and hesperetin-PC complexes may be prepared by the solvent dispersion technique.

Preparation in brief: Hesperetin (5 mg) and TPGS or hesperetin and PC at different weight ratios (1:3, 1:6, 1:9, 1:12, and 1:15) may be dissolved in 10 mL of methanol by ultrasound in a 50-mL round bottom flask. After ultrasonic homogenization, the mixture may be stirred for 30 min at 40°C and methanol is evaporated using a rotary evaporator at a negative pressure of 0.095 MPa. The final product may be dried under vacuum (50 Pa) to remove traces of solvents and kept in a glass desiccator.

More information may be obtained from the article attached below.

More information on solubility of hesperetin in various solvents may be found in the articles attached below.

Best.

Hello Priti Panchal

Nitrite is determined through formation of a reddish purple azo dye produced at pH 2.0 to 2.5 by coupling diazotized sulfanilamide with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED dihydrochloride). In other words, the sample containing nitrite is reacted with Sulfanilimide and N-(1-naphthyl)-ethylenediamine to form a colored species that absorbs at 540 nm.

This Griess Reagent System is based on a chemical reaction that uses sulfanilamide and N-1-naphthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. So, please check the pH.

DMEM (high glucose) is buffered for high CO2, and sodium bicarbonate in DMEM can cause the pH to become basic if CO2 is removed.

Phenol red does not interfere with Griess reaction. It will interfere with the absorbance reading taken at 540nm. It is therefore recommended to use media without phenol red to avoid any interference in the absorbance readings.

Best.

Dear Malcolm Nobre thank u for your contribution.

The Anti-Adherence Rinsing Solution is effective when applied to any plate type, not just AggreWell™ plates. However, its performance is not optimal on tissue culture-treated plates and you may see failures after a couple of days. 

Our Product and Scientific Support Team would be happy to work with you directly and troubleshoot this issue—you can email them at techsupport@stemcell.com.

Hi,

Here are a few solutions I hope they will work:

1. Reduce the trypsin exposure time. Instead of 2-3 minutes, try monitoring under a microscope and stop as soon as the cells detach (usually under 1-2 minutes).

2. Avoid directly placing thawed cells in T75 flasks. Instead, seed them at a higher density in a smaller vessel (e.g., T25 or T12.5) for the first passage to encourage better recovery.

3. Perform a mycoplasma test to ensure your culture is clean.

4. Try obtaining a fresh vial of PC3 cells from the ATCC or another reliable source.

5. Try changing the media more frequently (e.g., daily instead of every 2 days).

6. Ensure media preparation and storage conditions.

Yes, we are also cultivating neuroprogenitor cells and neurons and have recently encountered a similar issue. Unfortunately, We observed significant cell loss, aand even when we attempted to expand them, they do not survive. Like you, we verified key parameters, but found no abnormalities. Interestingly, other cell types that don't use B27 and N2 do not seem to exhibit this problem.

Hello Shahad Alsharif

There could be various reasons.

1. If the cell suspension is not mixed adequately before addition to the culture vessel, it could result in uneven distribution, attachment, and growth of cells. You may add the media with cells and gently tilt the vessel in an appropriate manner which would result in an effective way of mixing medium thoroughly without creating bubbles or foam.

2. Uneven growth can occur because of the shear forces generated by medium sweeping across the cell monolayer during medium changes or while moving cultures between the laminar flow hood and the incubator.

3. Unusual patterns of growth in the culture vessel could also occur when the vessel in the incubator is not at a leveled base. If you use shelves in the incubator that have not been leveled prior to use, it may often cause this effect. The shelves in the incubator should be checked with a spirit level and adjustments should be made following the incubator manufacturer’s recommendations. You should periodically check and make sure that the shelves are leveled, especially when they are removed for cleaning purposes.

4. Vibrations can cause unusual cell growth. Vibrations can come from a loose fan motor in the incubator or from any other source such as the motorized appliances. Keep incubators on sturdy surfaces that don't have any other pieces of equipment that vibrate.

5. Temperature differences within the incubator may create such a problem even when the differences are only a few tenths of a degree. For instance, cells prefer the warmer areas which are directly over the metal portion of the shelf. These conditions usually occur when incubators are frequently opened, especially during the first few hours after freshly inoculated cultures are placed inside the incubator.

On the other hand, if the temperature in the incubator is a bit too high for the cells, then the cells would prefer the holes in the shelf for cell growth and attachment. This clearly shows that cells are sensitive to very small temperature changes.

Maybe one of the above could be the cause of the problem. Please check!

Best.

Currently I have bleached and scrapped the infested cultures, but just need to know what they are to take caution accordingly